Clear after ETC but turbid after PBST washing

I would like to share my current experience on clearing mouse brain. I was also wondering what are your thoughts on these points?

1. After clearing mouse brain (23V, 1.03A, 37C), I can easily look through when holding the sample against daylight. However, when I continue following the protocol with washing the sample in PBST (24hrs x 2), the tissue turns turbid (which resolves after mounting).

2. I usually mount my sample in 80% glycerol to see if the clearing was sufficient and only for imaging in focusclear. The mounted tissue usually becomes pretty translucent. However, my last sample was very clear at the first couple of hours. When I looked at it the next day, it almost seemed like it has never been cleared at all.

Comments

1. Do you wash at room temperature or in 4 d.C.? Since in low temperatures, SDS is crystallised which might explain the turbid tissue, even when you mount it at RT later.2. The same could have happened for this point.. I'd try washing the tissue more thorough at RT.

I don't know about the glycerol issue since we usually place our samples in FocusClear immediately, but it is definitely normal for the sample to turn more opaque in PBST than it appears in either the clearing or mounting solution. This can probably be explained by refractive index differences between the different solutions which affects how transparent the sample appears.

That sample looks very yellow. Are you getting a lot of autofluorescence? I've been passively clearing my mouse brains. The brains don't turn yellow and autofluorescence is almost nonexistent. (A word of caution: I passively cleared some brains for so long that they were completely transparent and the consistency of jelly. The label -- Thy1 EGFP, transgenic -- all but disappeared. Don't let your brains go that long.)

It's true, this sample looks very yellow because I used the hardest conditions I could in the ETC. It had been passively clearing for one month (I had other samples in the ETC) and was in ETC at 50ºC and ~40V (2A) for 24h. These values are too high, burning ("yellowishing" the sample) only in one day. And yes, more burning implies more autofluorescence.

Glad this info is on here. I just put my first batch of cleared brains in PBST last night. I came in to find them looking whitish, and I thought I remembered this being a possibility. We'll see what happens when I mount them. I just wish these had endogenous GFP instead of being practice brains from CD1 pups we didn't need.